Anthrax Lethal Factor (LF) is a metalloproteinase introduced into the cells of mammalian hosts during infection by the bacterium Bacillus anthracis. LF disrupts intracellular signaling pathways mediated by the MAPKKs, resulting in cell toxicity, sepsis, respiratory failure and rapid death. While antibiotic therapy is effective if given in the first 48 h after symptoms appear, it cannot prevent death if initiated after this narrow therapeutic window. Although exposure to this pathogen is limited in nature, the lethality of anthrax spores as a bioterrorism weapon was demonstrated in the 2001 attacks through the US mail. Novel therapeutics capable of inhibiting the toxic effects of LF are needed to combat late-stage disease. Current treatment methods such as antibiotics, vaccines, and biologic therapies in development, suffer from significant drawbacks. Common to each is an inability to protect cells from the direct effects of LF. These therapies are also vulnerable to next generation genetically modified weapons exploiting LF toxicity. Directly targeting LF activity may allow synergy with current therapies and is the only proposed method of anthrax therapy that may provide rescue during late-stage infection. Use of an LF inhibitor has recently been validated in animals as a therapeutic approach. To date, however, no reported LF inhibitors possess the characteristics needed for a drug capable of protecting the general population in the event of a large scale bioterrorism anthrax attack. The current strategy at Hawaii Biotech Inc. (HBI) employs a three tiered screening cascade using assays designed to identify potent LF inhibitors with good PK properties and efficacy in animal models of anthrax infection. Achievement of this goal will require the continued medicinal chemistry optimization of inhibitors discovered at HBI as well as the identification of new lead series. Computational high throughput docking protocols developed at HBI and tailored specifically towards LF will be used to discover new inhibitors as backups to our current leads. In vitro profiling of compounds to assess potency and selectivity for LF along with molecular modeling of lead series will guide potency optimization. A unique panel of LF mutants prepared at HBI will also be used to guide the design and optimization of novel LF inhibitors. The physicochemical properties, PK, and ADMET characteristics of potent inhibitors will be determined to allow selection of the best candidates for in vivo testing. Finally, the efficacy of potential pre-clinical candidates will be demonstrated in established animal models of anthrax infection. The work proposed in this Phase II Renewal will build on HBI's expert knowledge of LF as a drug target with the goal of identifying an orally bioavailable preclinical candidate that demonstrates in vivo efficacy in a post-exposure animal model of anthrax lethal factor intoxication. The 2001 bioterrorism attack which delivered anthrax spores through the US mail resulted in eleven confirmed cases of the disease and led to five deaths. The current lack of an effective treatment for exposure to anthrax makes a repeat this unfortunate tragedy a real possibility. The goal of this research is to identify a drug which will protect the general public from any future acts of bioterrorism using anthrax as a weapon.